Establishment of an effective and uniform vaccine development strategy such as we propose here is key to conquering current and emerging infectious diseases. Despite successes against an array of bacterial agents, current approaches to vaccine development are as diverse as the microbes they target and require adjuvants that often have limited efficacy and/or toxic side effects. As a consequence, vaccine discovery is often slow, inefficient, and unsuccessful in the case of many high priority pathogens. We propose/hypothesize that vaccine generation for bacterial pathogens can be unified and stream-lined through a highly innovative criteria-directed approach that maximizes three of the most important criteria for vaccine success: Antigen (Ag)-mimicry, Adjuvanticity, and Efficient targeting of immunogens to Antigen Presenting Cells (APCs). We will test our hypothesis in the context of a very stringent model (respiratory infection with human-virulent Francisella tularensis [Ft]) in which we have obtained highly encouraging results with an adjuvant-free, mucosal vaccine (up to 75% protection of C57BL/6 mice). In fact, this is the only instance thus far in which it has been demonstrated that inactivated Ft administered intranasally protects C57BL/6 mice against mucosal challenge with the highly virulent Category A Ft SchuS4 pathogen. Thus, we will use Ft SchuS4 as a model pathogen to establish a criteria-directed approach to vaccine development, which we strongly believe will result in a fully protective adjuvant-independent mucosal vaccine against Ft SchuS4. In Aim 1, we will use differential cultivation and genetic manipulation of Ft SchuS4 to modulate: i) the antigenic similarity between in vitro- cultivated Ft immunogen and the replicating infectious pathogen (host-adaptation), ii) the cell-stimulatory capacity of the immunogen via over-expression of TLR2 agonist, and iii) processing/presentation of immunogen via bacterial surface expression of C3d, a ligand for CR2 on APCs (B cells and follicular dendritic cells). In Aim 2, we will analyze the impact of: i) growt medium-induced host-adaptation, and ii) over- expression of TLR2 ligands on TLR2 signaling, as reflected in dendritic cell (DC) maturation and cytokine responses. The impact of antigenic host-adaptation and altered TLR2 signaling on the processing/presentation of immunogen to Ft-specific T cells by DCs will also be evaluated. In Aim 3, we will: i) determine and optimize the protective activity of immunogens generated in Aims 1-2, separately and in combination, and ii) optimize the efficiency of Ft immunogen processing/presentation via the targeting of immunogen to CR2 and/or Fc receptors on APCs. The most protective immunogen(s) will then be evaluated in wildtype and humanized mice via aerosol challenge and correlates/mechanisms of protection identified. This project will not only yield an adjuvant-free mucosal vaccine against a Category A biothreat agent, but will establish an innovative and unique criteria-directed approach/platform for vaccine development applicable to other infectious agents, thus profoundly impacting preventive medicine/public health and changing the paradigm for vaccine development.